Antibodies are specific immunoglobulin (Ig) polypeptides produced by the vertebrate immune system in response to challenges by foreign proteins, glycoproteins, cells, or other antigenic foreign substances. The binding specificity of such polypeptides to a particular antigen is highly refined, with each antibody being almost exclusively directed to the particular antigen which elicited it. This specificity resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions do influence the overall domain structure and hence the combining site.
There are two major methods for generating vertebrate antibodies: generation of polyclonal antibodies in situ by mammalian B lymphocytes and generation of monoclonal antibodies in cell culture by B cell hybrids.
To generate antibodies in situ, an animal (such as a mouse or rabbit) is injected with an antigen. Several weeks later, blood is drawn from the animal and centrifuged. The resulting serum contains antibodies against the injected antigen. The resulting antibodies are polyclonal antibodies because they are products of many different populations of antibody producing cells and hence differ somewhat in their precise specificity and affinity for the antigen.
Monoclonal antibodies are produced using hybridoma technology in which an antibody producing cell is fused with a tumor cell that has the capacity for unlimited proliferation. In contrast to polyclonal antibodies, monoclonal antibodies are homogeneous because they are synthesized by a population of identical cells that are derived from a single hybridoma cell.
However, the use of monoclonal antibodies in humans is severely restricted when the monoclonal antibody is produced in a non-human animal. Repeated injections in humans of a “foreign” antibody, such as a mouse antibody, may lead to harmful hypersensitivity reactions, i.e., anti-mouse antibody (HAMA) or an anti-idiotypic, response. The HAMA response makes repeated administrations ineffective due to an increased rate of clearance from the patient's serum and/or allergic reactions by the patient.
Attempts have been made to manufacture human-derived monoclonal antibodies using human hybridomas. Unfortunately, yields of monoclonal antibodies from human hybridoma cell lines are relatively low compared to mouse hybridomas. Additionally, human cell lines expressing immunoglobulins are relatively unstable compared to mouse cell lines, and the antibody producing capability of these human cell lines is transient. Thus, while human immunoglobulins are highly desirable, human hybridoma techniques have not yet reached the stage where human monoclonal antibodies with the required antigenic specificities can be easily obtained.
Thus, antibodies of non-human origin have been genetically engineered to create chimeric or humanized antibodies. Such genetic engineering results in antibodies with a reduced risk of a HAMA response compared to that expected after injection of a human patient with a mouse antibody. For example, chimeric antibodies can be formed by grafting non-human variable regions to human constant regions. Khazaeli et al. (1991), J. Immunotherapy 15:42-52. Generally humanized antibodies, are formed by grafting non-human complementarity determining regions (CDRs) onto human framework regions (FRs) (See European Patent Application 0 239 400; Jones et al. (1986), Nature (London), 321:522-525; and Reichman et al. (1988), Nature (London), 332:323-327). Typically, humanized monoclonal antibodies are formed by grafting all six (three light chain and three heavy chain) CDRs from a non-human antibody into Framework Regions (FRs) of a human antibody. Alternately, Fv antibodies (See U.S. Pat. No. 4,642,334) or single chain Fv (SCFV) antibodies (See U.S. Pat. No. 4,946,778) can be employed to reduce the risk of a HAMA response.
However, these modified antibodies still retain various non-human light and heavy chain variable regions: the chimeric, Fv and single chain Fv antibodies retain entire non-human variable regions and CDR-grafted antibodies retain CDR of non-human origin. Such non-human regions can elicit an immunogenic reaction when administered to a human patient. Thus, many humanized MAbs remain immunogenic in both subhuman primates and in humans, with the humoral response of the host directed towards the variable region of these MAb (Hakimi et al. (1991), J. Immunol., 147:1352-1359; Stephens et al. (1995), Immunology, 85:668-674; Singer et al. (1993), J. Immunol., 150:2844-2857; and Sharkey et al. (1995), Cancer Res. 55:5935s-5945s).
One known human carcinoma tumor antigen is tumor associated glycoprotein-72 (TAG-72), as defined by monoclonal antibody B72.3 (See Thor et al., (1986) Cancer Res., 46:3118-3124; and Johnson et al., (1986), Cancer Res., 46:850-85). TAG-72 is associated with the surface of certain tumor cells of human origin.
Numerous murine monoclonal antibodies have been developed which have binding specificity for TAG-72. Exemplary murine monoclonal antibodies include the “CC” (colon cancer) monoclonal antibodies, which are a library of murine monoclonal antibodies developed using TAG-72. Certain CC antibodies have been deposited with the ATCC, including CC49 (ATCC No. HB 9459). Monoclonal antibody (MAb) CC49 is a second-generation antibody of B72.3 that reacts with the pancarcinoma tumor-associated antigen, TAG-72. Radiolabeled MAb CC49 has been shown to target tumor in both animal models and in ongoing radioimmunotherapeutic and raiodimmunodiagnostic clinical trials. (Divgi et al. (1994) Nucl. Med. Biol., 21:9-15; Meredith et al. (1994), J. Nucl. Med., 35:1017-1022; Mulligan et al. (1995), Clin. Cancer Res., 1:1447-1454; Arnold et al. (1992), Ann. Surgery, 216:627-632)The potential clinical utility of MAb CC49 is evident both from animal studies and ongoing clinical trials with the antibody. However, patients administered MAb CC49 do generate HAMA responses (Divgi et al, (1994) Nuc. Med. Biol., 21:9-15); Mulligan et al., (1995) Clin. Cancer Res., 1:1447-1454).
A humanized monoclonal antibody (HuCC49) has been formed by grafting hypervariable regions from monoclonal antibody CC49 into variable light (VL) and variable heavy (VH) frameworks of human monoclonal antibodies LEN and 21/28′CL, respectively, while retaining murine framework residues required for integrity of the antigen combining-site structure. (See, Kashmiri et al., (1995) Hybridoma, 14(5):461-473). This HuCC49 was shown to bind the TAG-72 antigen, albeit with a lower affinity, and demonstrated equivalent tumor targeting in animal models bearing human tumor xenografts.
It has been shown that not all residues of CDRs are critical in the complementarity of antigen/antibody surfaces. Known structures of the antigen-antibody complexes suggests that only 20-33% of CDR residues are involved in antigen contact (Padlan, (1994) Mol. Immunol., 31: 169-217). A comprehensive analysis of the available data of the sequences and the three dimensional structure of antibody combining sites has helped identify CDR residues that may be most critical in the antigen antibody interaction (Padlan et al., (1995) FASEB J., 9:133-139). These residues are designated as specificity determining residues (SDRs). Specificity determining residues vary between antibodies.